Oral Treatment with Lactococcus lactis Expressing Staphylococcus hyicus Lipase Enhances Lipid Digestion in Pigs with Induced Pancreatic Insufficiency

The Staphylococcus hyicus lip gene was cloned in Lactococcuslactis. Pancreatic insufficiency was induced by ligation ofthe pancreatic duct in pigs. In pigs who had undergone pancreaticligation, the coefficient of fat absorption was higher afterconsumption of lipase-expressing L. lactis (91.9% ± 3.7%)than that after consumption of the inactive control strain (78.4%± 2.4%).

INTRODUCTION

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Introduction

The efficiency of pancreatic enzyme formulations used to treatpancreatic insufficiency is often unsatisfactory. Researchersare therefore trying to find new sources of lipases and newdelivery vehicles (10). It was shown in a previous study thatLactococcus lactis was able to secrete the bacterial lipaseof Staphylococcus hyicus (5). The aim of this study was to determinethe effectiveness of genetically engineered L. lactis cellsexpressing the bacterial lipase of S. hyicus to help lipid digestionin pigs with induced pancreatic insufficiency.

L. lactis and thermoresistant spores of Bacillus subtilis weregrown and enumerated as previously described (3, 6). Chloramphenicoland erythromycin (Merck) at a concentration of 10 µg/ml,rifampin at 50 µg/ml, streptomycin (Research OrganicsInc.) at 1,000 µg/ml, and nisin powder (2.5% nisin content;ICN International) at 0.5 µg/ml were used. Basic methodsfor L. lactis genetic modification have been described previously(5). The L. lactis subsp. cremoris strain NZ9000 used in thisstudy was derived from strain MG1363 and contained the genespepN::nisRnisK (4). To obtain streptomycin- and rifampin-resistantL. lactis (strains JIM7017 and JIM7600), the strains were platedsuccessively onto M17 agar containing 1,000 µg of streptomycin/mland then onto agar containing 50 µg of rifampin/ml. StrainJIM7017 was the control strain, devoid of the lip gene.

Expression of the S. hyicus lip gene in L. lactis.

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Expression of the s....

pJIM2093 (5) was amplified by PCR with the oligonucleotides5'-AATCTTTTGTACCC ATGGTGAGTGCCTC-3' and 5'-GGGGGCGTGGCAGGCCATG GATTCG-3'. The bases in boldface indicate the mutationsinserted in order to create an NcoI site (in italics) and todelete the signal peptide encoding sequence. The PCR fragmentwas digested by NcoI and ligated. The resulting plasmid, pJIM2095,carried the lip gene without its signal peptide encoding sequence.It was transformed in L. lactis NZ9000 (4) to yield JIM7600.

Lipase expression was induced with a nisin concentration optimalfor heterologous protein expression in L. lactis (5). An overnightculture was used to inoculate fresh M17 medium. When an opticaldensity at 600 nm of 0.3 was reached, we added the nisin andfollowed lipase expression in the cell extracts by Western blottingof the proteins (carried out as previously described [8]) withan anti-lipase serum (5) (Fig. 1). The lipase production wasmaximal 2 h after nisin induction and was still stable 5 h afterinduction. We found that lipase represented about 15% of theproteins of the cell (data not shown; scanning gel stained withCoomassie blue).

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FIG. 1. Western blot of total cell extract proteins from the lactococcal strain expressing the S. hyicus lipase. Samples were loaded at different times after nisin induction (in minutes).

Pancreatic duct ligation.

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Pancreatic duct ligation.

To perform pancreatic duct ligation (PDL), pigs were deprivedof food for 24 h before surgery. After a right lateral laparotomyunder halothane anesthesia, two ligatures were placed closeto each other around the pancreatic duct at about 0.5 cm fromthe duodenum (1). The duct was then cut between the ligatures.After surgery, the pigs were housed in individual cages andtheir dietary intake was progressively restored to two dailymeals of 800 g each (300 g of food mixed with 500 ml of water)at 10 am and 4 pm.

Fate of L. lactis in the digestive tract.

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Fate of l. lactis...

Conventional pigs as well as the pigs with PDL receive 500 mlof a concentrated culture of L. lactis prepared as follows.A 3-liter overnight culture was centrifuged for 10 min at 6,000x g at 30°C. The bacterial pellet was resuspended in 500ml of fresh M17 medium mixed with B. subtilis spores and 1.5mg of nisin. The final mixture obtained, mixed with the powdereddiet, contained 9.4 x 10⁹ ± 0.4 L. lactis cells/g and9.4 x 10⁹ ± 0.3 B. subtilis spores/g. Two hours afterthe beginning of their meal, the animals were anesthetized withhalothane. After a right lateral laparotomy, each nonempty partof the small intestine containing the diet was ligated. Theduodenum was always empty. The contents of the intestine betweenthe jejunum and the ileum were removed. The stomach and cecumcontents were homogenized, and samples were collected. Beforeslaughtering the pigs, we verified that the pancreatic tissueof the pigs receiving ligation was atrophied and that the pancreaticduct was dilated. The fecal samples were weighed, homogenized,and diluted (1/10) in sterile water. L. lactis and B. subtiliscounts were measured from these dilutions for each sample. Pigswith or without PDL received the control L. lactis strain JIM7017(Fig. 2). The survival of L. lactis in the stomach and the upperparts of the jejunum was low for the two groups of pigs buthigher after PDL. In the mid-jejuno-ileum (parts 2 and 3), itssurvival did not differ between pigs with or without PDL.

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FIG. 2. Comparison between transit of B. subtilis spores and L. lactis cells in the digestive tracts of pigs with or without PDL. Grey symbols, pigs without PDL (n = 6); black symbols, pigs with PDL (n = 6); squares, thermoresistant spores of B. subtilis; triangles, L. lactis JIM7017; Inoc., inoculum of spores and L. lactis. The duodenum was always empty, and samples were not collected from it. The jejuno-ileum was divided into four equal-length segments, and the contents were collected. Vertical bars represent standard errors of the means.

Treatment of induced pancreatic insufficiency with L. lactis expressing the S. hyicus lip gene.

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Treatment of induced pancreatic...

The treatment of induced pancreatic insufficiency with L. lactisexpressing S. hyicus lip was carried out on four pigs with PDL(Table 1; PDL occurred at day zero). The pigs were adapted toreceive a high-fat diet (containing 15% lipids). To determinethe efficiency of L. lactis expressing the S. hyicus lipase(JIM7600), the four pigs with PDL were fed the high-fat dietas two daily meals of 300 g each mixed with 500 ml of a concentratedculture of L. lactis JIM7600 or a negative L. lactis control(JIM7017). These concentrated cultures were prepared as describedabove. The pigs served as their own controls. They receivedboth strains for 4 days each, interrupted by a 10-day washoutperiod (strains at days 21 to 24 and then at days 35 to 38).The stools were collected twice per day over each 4-day periodof bacterial treatment. Stools were immediately frozen. Fecaland dietary fats were measured (7), and the coefficient of fatabsorption (CFA) was calculated as the difference between ingestedand excreted fats, which yielded the percentage of ingestedfat (2). Results are expressed as means with their standarderrors. Values were compared by analysis of variance using SASsoftware. The CFA in normal pigs was 97% ± 2%. CFAs fromthe results obtained during the two treatment periods are shownin Table 1. The CFA was enhanced in all animals when the pigsingested the strain producing lipase (91.9% ± 3.7%) comparedto when they ingested the inactive strain (78.4% ± 2.4%),and the difference between both periods was significant (P <0.01).

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TABLE 1. CFA determined at different phases of the experimental schedule

The aim of the present study was to test the hypothesis thatlactic acid bacteria expressing an heterologous lipase wereefficient in reducing induced pancreatic insufficiency. We inducedexocrine pancreatic insufficiency in pigs through PDL. A recentexperimental study in dogs suggested that an acid- and protease-resistantlipase of bacterial origin might improve fat absorption (10,11). Since lactococci are rapidly destroyed by bile acids andproteolytic secretions (6, 9), we hypothesized that lactococcigenetically modified to have a high production of a bacteriallipase could be used as a vector instead of the pure enzyme.It was previously shown that S. hyicus lipase could be producedin L. lactis (5). In this study, we improved the lipase productionby removing the initial peptide signal encoding sequence necessaryfor secretion.

In pigs with or without PDL, the majority of ingested L. lactiscells lost their viability in the upper jejuno-ileum. It waspreviously shown that dead L. lactis released its cellular contentin the digestive tracts of mice (6). We have shown that in thefour pigs with PDL, L. lactis producing lipase could correctsteatorrhea when ingested with a high-lipid meal. The CFA showedsignificant improvement over the treatment period compared tothat obtained under the period of treatment with lipase-devoidL. lactis. We conclude that lactic acid bacteria, such as L.lactis modified to express lipase, could be used as a new vectorto deliver lipase in the gastrointestinal tract. This is potentiallya major advancement in the treatment of pancreatic insufficiencyin humans. The cost and safety of such a treatment also seemspromising.

ACKNOWLEDGMENTS

We are grateful to T. Corring for helpful advice, to C. Gibardfor lipid analyses in food and feces, to F. Cointpas and F.Gérard, to D. Besnard and M.-G. Brachet for pig surgeryand animal management, and to P. Rapine for technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Unité d'Ecologie et de Physiologie du Systeme digestif, Institut National de la Recherche Agronomique, Batiment 440, 78352 Jouy en Josas Cedex, France. Phone: 33-1 34 65 24 67. Fax: 33-1 34 65 24 62. E-mail: corthier{at}jouy.inra.fr.

REFERENCES

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